Polymer composite with internally distributed deposition matter

ABSTRACT

A process for the preparation of a polymer composite comprising internally distributed deposition matter wherein the process comprises providing a deposit of deposition matter at the surface of a solid state polymer substrate, contacting the surface deposited polymer with a plasticising fluid or a mixture of plasticising fluids under plasticising conditions to plasticise and/or swell the polymer and internally distribute deposition matter, and releasing the plasticising fluid or fluids to obtain polymer composite.; A polymer composite comprising a porous or non porous polymer throughout which particulate deposition matter as hereinbefore defined is distributed with desired uniformity, preferably with high uniformity in excess of 80% for example in excess of 98%.; A scaffold comprising a polymer composite having internally distributed deposition matter; and use of the composite as a support or scaffold for drug delivery, for use in bioremediation, as a biocatalyst or biobarrier for human or animal or plant matter, for use as a structural component, for example comprising the polymer and optional additional synthetic or natural metal, plastic, carbon or glass fibre mesh, scrim, rod or like reinforcing for medical or surgical insertion, for insertion as a solid monolith into bone or tissue, as fillers or cements for wet insertion into bone or teeth or as solid aggregates or monoliths for orthopaedic implants such as pins, or dental implants such as crowns etc.

The present invention relates to a process for the preparation of apolymer composite comprising contacting polymer with plasticising fluidand deposition matter and isolating polymer comprising internallydistributed deposition matter, the polymer composite obtained thereby,and apparatus for the preparation thereof, a polymer scaffold, drugdelivery device or the like comprising the composite in suitably sizedand shaped form, the use as a pharmaceutical or veterinary product, ahuman or animal health or growth promoting, structural, fragrance orcosmetic product, an agrochemical or crop protection product, inbiomedical, catalytic and like applications, more particularly as abiodegradable slow release product, or as biodegradable surgicalimplant, synthetic bone composite, organ module, and the like or forbioremediation, as a biocatalyst or biobarrier and the like.

The use of supercritical fluids in the production of polymers as aplasticising, foaming or purification agent is known. Supercriticalfluids (SCFs) act as plasticisers for many polymers, increasing themobility of the polymer chains. This results in an increase in the freevolume within the polymeric material.

Supercritical fluid has found application in incorporation of dyes andother inorganic materials which are insoluble in the supercriticalfluid, for example inorganic carbonates and oxides, into polymers with agood dispersion to improve quality, in particular dispersion in productssuch as paints for spray coating and the like.

Moreover the fluid can be used to foam the polymer by transition tonon-critical gaseous state whereby a porous material may be obtained andthis has been disclosed in U.S. Pat. No. 5,340,614, WO91/09079 & U.S.Pat. No. 4,598,006.

U.S. Pat. No. 5,340,614 discloses simultaneously contacting polymer,impregnation additive and SCF. U.S. Pat. No. 4,598,006 disclosesdissolving impregnation additive in SCF, adding polymer and releasingfluid with transition to subcritical conditions.

WO 91/09079 (De Ponti) discloses preloading polymer microspheres with anactive ingredient such as a drug by dissolving polymer in solvent,adding a solution of active ingredient, and mixing in silicone oil toobtain loaded microspheres. These are washed and hardened. Microspheresare then SCF processed to produce a porous structure.

However the double emulsion process of WO 91/09079 has shown in somecases only 68% retained drug activity compared with control and this isattributed to solvent effects, homogenising the double emulsion,breaking up droplets and the like.

Moreover this process is quite complex, requiring two polymer processingstages, and does not necessarily ensure good internal distribution.

Polymers have also been used in biomedical applications to developmaterials in which biocompatibility can be influenced to promotefavourable tissue responses whilst also producing materials withacceptable mechanical and surface properties. Biofunctional compositematerials e.g. calcium hydroxyapatite dispersed in various polymers arewell established for orthopaedic, dental and other applications. Thesematerials are prepared with very high loadings of inorganic solid, of upto 80%, in the form of a powder, and a composite is formed either byvigorous mixing of the powdered material into the solid or moltenpolymer, or by polymerisation of the monomers in the presence ofsuspended inorganic powders. In both cases, the material becomesentrapped within the polymer matrix.

These methods for preparation however are prone to insufficient anduncontrolled mixing of material leading to large aggregate formationwhereby the composite is prone to fracture and may not be suitable forcommercial processing.

WO 98/51347 (Howdle et al) discloses the preparation by dense phasefluid processing of biofunctional polymers comprising biofunctionalmaterial having the desired mechanical properties both for commercialprocessing and for implant into a human or animal host structure such asbone or cartilage, dental and tissue structures into which they aresurgically implanted for orthopaedic bone and implant, prosthetic,dental filling or restorative applications, prolonged releaseapplications and the like. Biofunctional material is in particular anypharmaceutical, veterinary, agrochemical, human and animal health andgrowth promoting, structural, cosmetic and toxin absorbing materials,such as a broad range of inorganic or organic molecules, peptides,proteins, enzymes, oligosaccharides, carbohydrates, nucleic acids andthe like.

Particular application is in the production of bone composites formedfrom a biofunctional polymer with inorganic calcium hydroxyapatiteuniformly distributed throughout. This process uses the addition of CO₂to plasticise polymeric material and highly efficient stirring to ensurehomogeneous incorporation of particulate material throughout thepolymer.

This and other work from the same authors has shown high uniformity.However there is a need for further improved uniformity for both highand low loading levels, with milder processing conditions. Therapeuticconcentrations of growth factors and other biotechnology drugs are ofthe order of ppb, whilst those of biocompatibilisers such ashydroxyapatite are of the order of 80 wt %. Greater uniformity manifestsitself in more uniform prolonged release, and stronger monolithicstructures.

We have now surprisingly found that controlled internal distribution ofmatter within a polymer composite can be achieved in a simple andreproducible process, which enables the accurate and efficient handlingof biologically active molecules in small or large amount in solutionwhile retaining the manifold advantages of SCF processing. The presentinvention provides deposition of matter on a polymer surface in a firststage and internal distribution and optional pore formation in a secondpolymer plasticisation stage. This is in contrast to WO 91/09079 whichteaches dissolving polymer and emulsifying with impregnation matter in afirst stage, and plasticising in a second stage.

Accordingly in the broadest aspect of the invention there is provided aprocess for the preparation of a polymer composite comprising internallydistributed deposition matter wherein the process comprises providing adeposit of deposition matter at the surface of a solid state polymersubstrate, contacting the surface deposited polymer with a plasticisingfluid, or a mixture of plasticising fluids under plasticising conditionsto plasticise and/or swell the polymer and internally distributedeposition matter, and releasing the plasticising fluid or fluids toobtain polymer composite.

Preferably the process comprises providing a deposit at the surface of ahigh surface area polymer substrate, more preferably a powder bed or ahigh porosity matrix. Preferably the process provides a deposition layerof deposition matter on the internal and external surfaces of thepolymer substrate, more preferably any exposed surfaces, including anyexposed surface pores. By this means a more dilute deposit is formedwhich is of greater uniformity than depositing the same quantity ofmaterial on a smaller surface area. Deposition may be over the entiresurface area or only part or parts thereof.

Preferably a porous solid state polymer substrate is obtained bycontacting polymer with plasticising fluid and subsequently releasingfluid in suitable manner to foam the polymer as is known in the art. Ina preferred embodiment therefore the process comprises in a first stagecontacting polymer with plasticising fluid or a mixture of plasticisingfluids under plasticising conditions to plasticise the polymer, andreleasing the fluid to obtain a solid state substrate polymer; in asecond stage providing a surface deposit of deposition matter at thesurface of the polymer, and in a third stage contacting the surfacedeposited polymer with a plasticising fluid or a mixture of plasticisingfluids under plasticising conditions to plasticise and/or swell thepolymer and internally distribute deposition matter, and releasing theplasticising fluid or fluids to obtain polymer composite. Preferably inthe first stage the plasticising and releasing the fluid(s) is in mannerto foam the polymer and obtain a porous solid state substrate polymer,for use in the second stage.

The product composite may be porous or non-porous, even if obtained froma porous substrate. It is a particular advantage that porosity may serveto facilitate surface deposition, but be of little interest in theproduct composite or vice versa or a combination thereof.

Deposition may be of discrete particles or of dissolved depositionmatter and may be by solid or fluid phase deposition. Preferablydeposition matter is provided in fluid phase, and deposition comprisesimmersion, spraying and the like with a solution, dispersion orsuspension of deposition matter and drying by freezing, evaporation,heating, blotting etc.

Alternatively deposition matter is provided in solid phase anddeposition comprises powder coating, dusting, rolling or adhering.

Deposition may be aided by softening or adhesion of surface polymer, inparticularly in the case of deposition of insoluble or dry phasedeposition matter.

Deposition may be with or without physical interaction with the polymersurface. In a particularly preferred embodiment, on contacting polymersubstrate with a solution, dispersion or suspension of depositionmatter, the deposition matter adsorbs from liquid phase onto the polymersurface and forms an adsorption layer of deposition matter at desiredlevels. This layer remains intact to solvent and impact effects and thelike, for example if subsequently surface washed with liquids.

Immersion time may be of the order 1 second up to 48 hours, depending onthe materials used. Drying time may be up to 48 hours depending onsensitivity to extreme heat or freezing or the like.

Preferably deposition matter is provided in particulate or powder formand may be of particle size in the range up to 1 mm, preferably 50-1000micron. Deposition matter may be of uniform or mixed particle size,depending on practical constraints and the required distribution, andmay be of same or different matter.

The polymer is suitably in the solid phase or is a highly viscous fluidand may present limited or good mixing characteristics. Solid phasepolymer may be particulate, eg in the form of granules, pellets,microspheres, powder, or monolithic eg matrix form. Plasticisingconditions comprise conditions of reduced viscosity to plasticise and/orswell the polymer. It is known that particulate polymer agglomerates onplasticisation to a larger structure. This may revert to a particulatecomposite or form a monolithic composite on release of plasticisingfluid, as hereinbelow defined. Polymer volumes of 5 or 10 mg or g up tomulti kg scale may be used.

Reference herein to a plasticising fluid is to a fluid which is able toplasticise polymer in its natural state or in supercritical, nearcritical, dense phase or subcritical state. Fluid may be liquid orgaseous, and is preferably selected for a suitable density which iscapable of plasticising a given polymer, fluid density may be in therange 0.001 g/ml up to 10 g/ml for example 0.001 g/ml up to 2 g/ml.

Plasticising conditions comprises elevated or ambient temperature,and/or elevated or ambient pressure. Fluid may be selected for effectiveplasticisation of a given polymer under conditions which are amenable tothe deposition matter or alternatively fluid is selected by preferredchemical type and suitable plasticising conditions are chosen for thatfluid, preferably selecting a first amenable condition (T) andcompensating with second condition (P) to obtain desired density.

Preferably the plasticising conditions comprise a desired temperatureless than, equal to or greater than the fluids critical temperature (Tc)in the range −200° C. to +500° C., preferably −200° C. to 200° C., morepreferably −100 to +100° C., for example −80 or −20° C. to +200 or +100°C. For most fluids this will be in the range approximately 10 to 15° C.,15 to 25° C., 25 to 30° C., 30 to 35° C., 35 to 45° C. or 45 to 55° C.,most preferably approximately 28 to 33° C. (CO₂). Other sub ranges maybe envisaged and are within the scope of the invention. Preferably thelowest temperature is employed which is compatible with sufficientlowering of the polymer Tg to achieve plasticisation. To operate atambient temperature, the process of the invention may requirecompensation by increase in pressure.

Preferably the plasticising fluid comprises a desired pressure lessthan, equal to or greater than the plasticising fluids critical pressure(Pc) from in excess of 1 bar to 10000 bar, preferably 1 to 1000, morepreferably 2 to 800 bar, more preferably 2 to 400 bar, more preferably 5to 265 bar, most preferably 15 to 75 bar. For most fluids this will bein the range approximately 30 to 40 bar, 40 to 50 bar, 50 to 60 bar, 60to 75 bar or 80 to 215 bar, and is most preferably approximately 34 to75 bar for dense phase or supercritical CO₂. Other sub ranges may beenvisaged and are within the scope of this invention.

Fluid may be provided at plasticising conditions prior to contactingwith polymer and deposition matter or may be brought to plasticisingconditions in contact with surface deposited polymer.

Preferably the process is carried out for a contact time of surfacedeposited polymer and plasticising fluid of 1 millisecond up to 5 hours.Short contact time may be preferred for example 2 milliseconds up to 10minutes, more preferably 20 milliseconds to 5 minutes, more preferably 1second to 1 minute, more preferably 2 to 30 seconds, most preferably 2to 15 seconds. Alternatively long contact time minimises detrimentaleffects of pressurising the vessel, and allows superior distribution,for example 15 minutes to 2 hours, preferably 15 minutes to 40 minutesor 30 minutes to 1 hour.

Pressurising plasticising fluid may be in situ, or ex situ prior tocontacting with surface deposited polymer as hereinbefore defined. Thepressurisation period whereby in the case of in situ or ex situpressurisation the fluid is pressurised or is introduced to the surfacedeposited polymer, is suitably for a period of 1 second to 3 minutes,more preferably from 1 second to 1 minute, more preferably from 1 to 45seconds.

The process may be carried out with or without stirring or blending.Blending and conditions may be selected to assist plasticisation oraccording to the desired uniformity and distribution of loading. In thecase that uniform distribution is required the process preferablycomprises blending for prolonged period and/or high intensity. In thecase that non-uniform distribution is envisaged, the process may becarried out simply with stirring.

Blending may be by physical mixing, pumping, agitation for example withaeration or fluidising gas flow, lamellar flow or otherwise impregnationor diffusion of plasticising fluid throughout the surface depositedpolymer. Stirring is typically with use of stirrers and impellers,preferably helical impellers such as helical ribbon impellers forenhanced blending and the like.

Blending may be for a period of 1 millisecond up to 5 hours and may befor the duration of contacting with plasticising fluid or otherwise.Preferably stirring or blending is for substantially the duration ofcontacting with plasticising fluid, with period of stirring or blendingcorresponding to period of plasticising fluid contacting as hereinbeforedefined.

The process comprises subsequently releasing the plasticising fluid. Inthe case that plasticising conditions comprises elevated pressurerelease is under reduced pressure conditions, conducted over a desireddepressurisation period, whereby the polymer composite is obtainedcomprising internally distributed deposition matter. Depressurisationmay be achieved in situ, by depressurising a pressure vessel in whichthe process is carried out, whereby a monolithic block of polymercomposite is obtained. Alternatively the contents of a pressure vesselin which the process is conducted may be discharged into a secondpressure vessel at lower pressure whereby a homogeneous powder ofpolymer composite as hereinbefore defined is obtained by known means.

Release of fluid may be in manner to foam the polymer substrate andcreate a porous structure, with deposition matter distributed throughoutthe polymer matrix and internal pore surface. Typically this is achievedby rapid release over a period of up to 2 minutes.

Depressurisation period may be selected to foam the polymer if desired,and therefore determines the porosity of composite. Transition ispreferably rapid over a period of from 1 ms to 10 minutes, preferablyfrom 1 second to 3 minutes, more preferably from 1 to 3 seconds for highporosity polymer. Alternatively plasticising fluid may be released inmanner to allow fluid diffusion out of the polymer, avoiding foaming, tocreate a non-porous structure. Typically this is achieved by prolongedgradual release of fluid over a period of in excess of 10 minutes up to12 hours. Preferably transition is to near ambient pressure i.e.substantially 1 atm (101.325 kPa).

The process may be carried out in the presence or absence of additionalsolvents or fluids. In the case of physical interaction of depositionmatter with the polymer surface additional solvents or fluids may beused without affecting the uniform deposition layer. Preferably howeverthe process is carried out in the absence of solvent capable ofdissolving the deposition matter. Suitable carriers, agents,preservation agents and the like may be employed as desired.

A plasticising fluid as hereinbefore defined may comprise any fluidwhich is capable of plasticising a desired polymer. As is known in theart such fluids may be subjected to conditions of elevated temperatureand pressure increasing density thereof up to and beyond a criticalpoint at which the equilibrium line between liquid and vapour regionsdisappears. Supercritical and dense phase fluids are characterised byproperties which are both gas like and liquid like. In particular, thefluid density and solubility properties resemble those of liquids,whilst the viscosity, surface tension and fluid diffusion rate in anymedium resemble those of a gas, giving gas like penetration of themedium.

Preferred plasticising fluids include carbon dioxide, di-nitrogen oxide,carbon disulphide, aliphatic C₂₋₁₀ hydrocarbons such as ethane, propane,butane, pentane, hexane, ethylene, and halogenated derivatives thereofsuch as for example carbon tetrafluoride or chloride and carbonmonochloride trifluoride, and fluoroform or chloroform, C₆₋₁₀ aromaticssuch as benzene, toluene and xylene, C₁₋₃ alcohols such as methanol andethanol, sulphur halides such as sulphur hexafluoride, ammonia, xenon,krypton and the like, and mixtures thereof. Typically these fluids maybe brought into plasticising conditions at temperature of between −200°C. to +500° C. and pressures of in excess of 1 bar to 10000 bar, ashereinbefore defined. It will be appreciated that the choice of fluidmay be made according to its properties, for example diffusion andpolymer plasticisation. Preferably the fluid acts as solvent forresidual components of a polymer composite as hereinbefore defined butnot for polymer or deposition matter as hereinbefore defined. Choice offluid may also be made with regard to critical conditions whichfacilitate the commercial preparation of the polymer as hereinbeforedefined. Supercritical conditions are shown of some fluids in Table 1.Fluid Critical Temperature/° C. Critical Pressure/bar Carbon dioxide31.1 73.8 Ethane 32.4 48.1 Ethylene 9.3 49.7 Nitrous oxide 36.6 71.4Xenon 16.7 57.6 Fluoroform CHF₃ 26.3 48.0 Monofluoromethane 42 55.3Tetrafluoroethane 55 40.6 Sulphur hexafluoride 45.7 37.1Chlorofluoromethane 29 38.2 Chlorotrifluoromethane 28.9 38.7 Nitrogen−147 33.9 Ammonia 132.5 111.3 Cyclohexane 280.3 40.2 Benzene 289.0 48.3Toluene 318.6 40.6 Trichlorofluoromethane 198.1 43.5 Propane 96.7 41.9Propylene 91.9 45.6 Isopropanol 235.2 47.0 p-xylene 343.1 34.7

Preferably the plasticising fluid comprises carbon dioxide optionally inadmixture with any further fluids as hereinbefore defined or mixed withconventional solvents, so-called “modifiers”. CO₂ is generally approvedby regulatory bodies for medical applications, is chemically inert,leaves no residue and is freely available.

The plasticising fluid may be present in any effective amount withrespect to the polymer. Preferably the plasticising fluid is provided ata desired concentration in the reaction vessel to give a desiredplasticisation and/or swelling of polymer. Such range may be from 1% to200% of the polymer weight, e.g. with plasticising fluid in sufficientexcess to achieve 10% to 40% absorption with respect to polymer weight.

The deposition matter may be present in any effective amount withrespect to polymer. Typical values are therefore 1×10⁻¹² wt % to 99.9 wt%, preferably 0.01 or 0.1 to 99.0 wt %, more preferably greater than 0.5wt % or 1.0 wt % up to 50 wt %. In a particularly preferred embodimenttherefore the process is carried out in low volumes of the order ofpicogram and nanogram levels with respect to 5 g amounts of polymer. Forexample, presented as concentration of deposition matter on polymer, lowvolumes in the range 1×10¹ to 1×10³ ng/mg may be present, for example 50to 150 ng/mg. This is beneficial for most biologically active moleculessuch as enzymes or protein molecules because their therapeuticconcentrations are very low. For example: the therapeutic amount of thegrowth factor HGF (hepatocyte growth factor) required to provide atherapeutic response in liver cells during liver regeneration process intissue engineering is 10 ng/ml ((Tsubouchi, Niitani et al. 1991).

The deposition matter may be selected from any desired matter adapted toperform a function on a desired biolocus comprising or otherwiseassociated with living matter, and which may be bioactive, bioinert,biocidal or the like; and non-biofunctional material including dyes,additives and the like.

Preferably deposition matter is selected from a component, or precursor,derivative or analogue thereof, of a host structure into whichimplantation or incorporation is desired and preferably comprises matterintended for growth or repair, shielding, protection, modification ormodelling of a human, animal, plant or other living host structure forexample the skeleton, organs, dental structure and the like; to combatantagonists; for metabolism of poisons, toxins, waste and the like orfor synthesis of useful products by natural processes, forbioremediation, biosynthesis, biocatalysis or the like.

More specifically the deposition material includes but is not limited tothe following examples typically classed as (pharmaceutical) drugs andveterinary products; agrochemicals as pest and plant growth controlagents; human and animal health products; human and animal growthpromoting, structural, or cosmetic products including products intendedfor growth or repair or modelling of the skeleton, organs, dentalstructure and the like; absorbent biodeposition materials for poisons,toxins and the like.

Pharmaceuticals and veterinary products, i.e. drugs, may be defined asany pharmacologically active compounds that alter physiologicalprocesses with the aim of treating, preventing, curing, mitigating ordiagnosing a disease.

Drugs may be composed of inorganic or organic molecules, peptides,proteins, enzymes, oligosaccharides, carbohydrates, nucleic acids andthe like.

Drugs may include but not be limited to compounds acting to treat thefollowing:

Infections such as antiviral drugs, antibacterial drugs, antifungaldrugs, antiprotozal drugs, anthelmintics,

Cardiovascular system such as positive inotropic drugs, diuretics,anti-arrhythmic drugs, beta-adrenoceptor blocking drugs, calcium channelblockers, sympathomimetics, anticoagulants, antiplatelet drugs,fibrinolytic drugs, lipid-lowering drugs;

Gastro-intestinal system agents such as antacids, antispasmodics,ulcer-healing, drugs, anti-diarrhoeal drugs, laxatives, central nervoussystem, hypnotics and anxiolytics, antipsychotics, antidepressants,central nervous system stimulants, appetite suppressants, drugs used totreat nausea and vomiting, analgesics, antiepileptics, drugs used inparkinsonism, drugs used in substance dependence;

Malignant disease and immunosuppresion agents such as cytotoxic drugs,immune response modulators, sex hormones and antagonists of malignantdiseases;

Respiratory system agents such as bronchodilators, corticosteroids,cromoglycate and related therapy, antihistamines, respiratorystimulants, pulmonary surfactants, systemic nasal decongestants;

Musculoskeletal and joint diseases agents such as drugs used inrheumatic diseases, drugs used in neuromuscular disorders; and

Immunological Products and Vaccines.

Agrochemicals and crop protection products may be defined as any pest orplant growth control agents, plant disease control agents, soilimprovement agents and the like. For example pest growth control agentsinclude insecticides, miticides, rodenticides, molluscicides,slugicides, vermicides (nematodes, anthelmintics), soil fumigants, pestrepellants and attractants such as pheromones etc, chemical warfareagents, and biological control agents such as microorganisms, predatorsand natural products;

plant growth control agents include herbicides, weedicides, defoliants,dessicants, fruit drop and set controllers, rooting compounds, sproutinginhibitors, growth stimulants and retardants, moss and lichencontrollers and plant genetic controllers or agents;

plant disease control agents include fungicides, viricides, timberpreservatives and bactericides; and

soil improvement agents include fertilisers, trace metal additives,bacterial action control stimulants and soil consolidation agents.

The deposition matter may alternatively or additionally comprise anyfunction enhancing components, including naturally occurring orsynthetic otherwise modified growth promoters, biocompatibilisers,vitamins, proteins, glycoproteins, enzymes, nucleic acid, carbohydrates,minerals, nutrients, steroids, ceramics and the like and functioningmatter such as spores, viruses, mammalian, plant and bacterial cells.Preferred deposition matter includes growth factors selected frombiocompatibilisers, vitamins, proteins, glycoproteins, enzymes, nucleicacid, carbohydrates, minerals, nutrients, steroids, ceramics and thelike; in particular growth factors such as basic Fibroblastic GrowthFactor, acid Fibroblastic Growth Factor, Epidermal Growth Factor, HumanGrowth Factor, Insulin Like Growth Factor, Platelet Derived GrowthFactor, Nerve Growth Factor and Transforming Growth Factor and bonemorphogenetic proteins; antitumorals such as BCNU or 1,3-bis(2-chloroethyl)-1-nitrosourea, daunorubicin, doxorubicin, epirubicin,idarubicin, 4-demethoxydaunorubicin3′-desamine-3′-(3-cyano-4-morpholinyl)-doxorubicin,4-demethoxydaunorubicin-3′-desamine-3′-(2-methoxy-4-morpholinyl)-doxorubicin,etoposide and teniposide; hormones such as LHRH and LHRH analogues; andsteroideals for birth control and/or antitumoral action such asmedroxyprogesterone acetate or megestrol acetate; tricalcium phosphateor the class of apatite derivatives, for example calcium hydroxyapatitewhich functions as a bone or dental component and promotesbiocompatability, silicon which functions as a tissue modellingcomponent, and analogues, precursors or functional derivatives thereof,bioactive species such as collagen, bioglasses and bioceramics, otherminerals, hyaluran, polyethyleneoxide, CMC (carboxymethylcellulose),proteins, organic polymers, and the like and components adapted forincorporation as implants into meniscus, cartilage, tissue and the likeand preferably promote growth, modelling, enhancing or reinforcing ofcollagen, fibroblasts and other natural components of these hoststructures.

Absorbent deposition matter for poisons, toxins and the like may bedefined as any natural or synthetic products capable of immobilising byabsorption, interaction, reaction or otherwise of naturally occurring orartificially introduced poisons or toxins.

The deposition matter may be in any desired form suited for the functionto be performed, for example in solid, semi-solid such as thixotrope orgel form, semi-fluid or fluid such as paste or liquid form, and may bemiscible or immiscible but is insoluble in the polymer and plasticisingfluid, eg as a suspension. It may be convenient to adapt the depositionmatter form to render it in preferred form for processing and thefunction to be performed. The matter is preferably in the form of solidparticles having particle size selected according to the desiredapplication. Preferably particle size is of similar or of lesser orderto that of the polymer composite, and optionally of any pores,preferably 10⁻⁹ m-10⁻² m, for example of the order of picometers,nanometers, micrometers, millimetres or centimetres.

The polymer composite may be in desired form suitable for thehereinbefore mentioned uses. For application to living matter, thepolymer composite may be introduced as a dry or wet spray, powder,pellets, granules, monoliths and the like, comprising the depositionmaterial substrate in releasable manner by dissolution, evaporation orthe like, for example in the hereinbefore defined agrochemical,insecticidal and the like uses. For administration as a healthcare,pharmaceutical or the like composition to the human or animal body, thecomposition may be suitably formulated according to conventionalpractices.

For use as pharmaceutical and veterinary products fabricated using theinventive process composites may be in the form of creams, gels, syrups,pastes, sprays, solutions, suspensions, powders, microparticles,granules, pills, capsules, tablets, pellets, suppositories, pessaries,colloidal matrices, monoliths and boluses and the like, foradministration by topical, oral, rectal, parenteral, epicutaneous,mucosal, intravenous, intramuscular, intrarespiratory or like.

The composite may be non porous or porous, and may comprise open orclosed cell pores. Composite obtained with a very open porous structure,known as microcellular, is ideal for prolonged or staged release, forpharmaceutical and animal health etc applications as hereinbeforedefined, also for biomedical and biocatalytic applications for examplesupporting growth of blood vessels and collagen fibres throughout thematrix, and forming structures resembling bone, meniscus, cartilage,tissue and the like, and providing a structure for throughput ofsubstrate for biocatalysis and bioremediation and the like.

Non-porous, open or closed cell composite may be useful forbiodegradable staged or prolonged release delivery applications ofdeposition matter not requiring leaching in or out or other access.Release may be in vitro or in vivo and by parenteral, oral, intravenous,application or surgical for release proximal to the treatment locus, egin tissue tumor treatment, or hyperthermic bone tumor treatment.

A porous polymer composite may be obtained with uniform or variedporosity, preferably provides pores of at least two different orders ofmagnitude, for example of micro and macro type, each present in anamount of between 1 and 99% of the total void fraction of the polymercomposite.

Reference herein to micro and macro pores is therefore to be understoodto be respectively pores of any unit dimension and its corresponding10^(n) multiple. For example micro pores may be of the order of 10⁻⁽¹⁰⁻⁷⁾ m with respective macro pores of the order of 10 ⁻⁽⁷⁻⁵⁾ m,preferably 10 ⁻⁽⁸⁻⁷⁾ m and 10 ⁻⁽⁶⁻⁵⁾ m respectively, more preferably ofmicron and 10² micron order, for example 50 to 200 micron. The pores maybe of any desired configuration. Preferably the pores form a network oftortuous interlinking channels, more preferably wherein the micro poresinterlink between the macro pores.

Deposition matter may be distributed throughout relatively smaller andrelatively larger pores or confined to larger pores. Deposition mattermay be embedded in the walls of pores or may be freely supported but notencased in polymer matrix.

An open cell structure may create a channel structure throughout thepolymer composite, for leaching in and out of fluids for prolongedrelease, or for supply and removal of materials, in particular fluidsand release matter. Different particle size deposition matter mayselectively distribute between smaller and larger pores.

A composite created in this manner may enhance the biomechanicalproperties of the polymer, in contrast to that of known polymerscomprising inhomogeneous distribution and large aggregates of inorganicmaterials.

The process may be controlled in manner to determine the dimensions andvoid fraction of micro and macro pores and the morphology of the finalproduct. The period for plasticising fluid release determines in partthe level of porosity. Additionally the difference in pressure isproportional to porosity. Also a higher critical temperature confers ahigher porosity. The composite is suitably obtained with porosity of 15%to 75% or greater, preferably 50% up to 97%.

Suitably the polymer retains its solid or highly viscous fluid formsubsequent to release of plasticising fluid, in order to retain theporous structure induced by the fluid.

Further processing of the polymer, for example additional extractionwith super critical fluid as known in the art or with other extractants,post-polymerisation and cross-linking, may be subsequently performed asrequired and as known in the art.

The polymer may be selected from any known polymer, (block) copolymer,mixtures and blends thereof which may be crosslinked or otherwise, whichis suited for introduction into or association with the human or animalbody, plants or other living matter, or in vitro, or for use in theenvironment in non-toxic manner. Suitable polymer materials are selectedfrom synthetic biodegradable polymers as disclosed in “PolymericBiomaterials” ed. Severian Dumitriu, ISBN 0-8247-8969-5, Publ. MarcelDekker, New York, USA, 1994, bioresorbable polymers syntheticnon-biodegradable polymers; and natural polymers. Preferably the polymeris selected from homopolymers, block and random copolymers, polymericblends and composites of monomers which may be straight chain, (hyper)branched or cross-linked.

Polymer may be of any molecular weight for the desired application, andis suitably in the range of from 1 to 1,000,000 repeat units. Highermolecular weight may be useful for longer release patterns or slowerdegradation.

Polymers may include but are not limited to the following which aregiven as illustration only.

Synthetic biodegradable polymers may be selected from:

Polyesters including poly(lactic acid), poly(glycolic acid), copolymersof lactic and glycolic acid, copolymers of lactic and glycolic acid withpoly(ethylene glycol), poly(e-caprolactone), poly(3-hydroxybutyrate),poly(p-dioxanone), poly(propylene fumarate);

Preferably polylactides include DD, DL, LL enantiomers and are preparedfrom D and L lactic acid and glycolic acid monomers, or a combinationthereof, or monomers such as 3-propiolactone tetramethylglycolide,b-butyrolactone, 4-butyrolactone, pivavolactone and intermolecularcyclic esters of alpha-hydroxy butyric acid, alpha-hydroxyisobutyricacid, alpha-hydroxyvaleric acid, alpha-hydroxyisovaleric acid,alpha-hydroxycaproic acid, alpha-hydroxy-alpha-ethylbutyric acid,alpha-hydroxyisocaproic acid, alpha-hydroxy-3-methylvaleric acid,alpha-hydroxyheptanoic acid, alpha-hydroxyoctanoic acid,alpha-hydroxydecanoic acid, alpha-hydroxymyristic acid,alpha-hydroxystearic acid, and alpha-hydroxylignoceric acid. It is mostpreferred to use lactic acid as sole monomer or lactic acid as theprincipal monomer with glycolic acid as the comonomer. The latter aretermed poly(lactide-co-glycolide) copolymers; particularly suitable arepolymers prepared from lactic acid alone, glycolic acid alone, or lacticacid and glycolic acid wherein the glycolic acid is present as acomonomer in a molar ratio of 100:0 to 40:60;

Poly (ortho esters) including Polyol/diketene acetals addition polymersas described by Heller in: ACS Symposium Series 567, 292-305, 1994;

Polyanhydrides including poly(sebacic anhydride) (PSA),poly(carboxybisbarboxyphenoxyphenoxyhexane) (PCPP),poly[bis(p-carboxyphenoxy) methane] (PCPM), copolymers of SA, CPP andCPM, as described by Tamada and Langer in Journal of BiomaterialsScience—Polymer Edition, 3, 315-353,1992 and by Domb in Chapter 8 of theHandbook of Biodegradable Polymers, ed. Domb A. J. and Wiseman R. M.,Harwood Academic Publishers;

Poly(amino acids); polyacetals; polyketals; polyorthoesters;

Poly(pseudo amino acids) including those described by James and Kohn inpages 389-403 of Controlled Drug Delivery Challenges and Strategies,American Chemical Society, Washington D.C.;

Polyphosphazenes including derivatives of poly[(dichloro) phosphazene],poly[(organo) phosphazenes], polymers described by Schacht inBiotechnology and Bioengineering, 52, 102-108, 1996; and

Azo Polymers

Including those described by Lloyd in International Journal ofPharmaceutics, 106, 255-260, 1994.

Synthetic Non-biodegradable Polymers may be selected from:

Vinyl polymers including polyethylene, poly(ethylene-co-vinyl acetate),polypropylene, poly(vinyl chloride), poly(vinyl acetate), poly(vinylalcohol) and copolymers of vinyl alcohol and vinyl acetate, poly(acrylicacid) poly(methacrylic acid), polyacrylamides, polymethacrylamides,polyacrylates, Poly(ethylene glycol), Poly(dimethyl siloxane),Polyurethanes, Polycarbonates, Polystyrene and derivatives.

Natural Polymers may be selected from carbohydrates, polypeptides andproteins including:

Starch, Cellulose and derivatives including ethylcellulose,methylcellulose, ethylhydroxyethylcellulose, sodiumcarboxymethylcellulose; Collagen; Gelatin; Dextran and derivatives;Alginates; Chitin; and Chitosan;

Preferably a non biodegradable polymer is selected from polymers such asester urethanes or epoxy, bis-maleimides, methacrylates such as methylor glycidyl methacrylate, tri-methylene carbonate, di-methylenetri-methylene carbonate; biodegradable synthetic polymers such asglycolic acid, glycolide, lactic acid, lactide, p-dioxanone,dioxepanone, alkylene oxalates and caprolactones such asgamma-caprolactone.

Polymer substrate may be obtained from its precursors in the process ofthe invention. The precursors may react to form the polymer substrate(s)in situ during or subsequent to plasticising fluid processing.

The polymer may comprise any additional polymeric components havingperformance enhancing or controlling effect, for example determining thedegree and nature of cross-linking for desired degradation, release, orfluid access, flexural and general mechanical properties, electricalproperties and the like.

Additional components which may be incorporated during the manufactureof the polymer composite, for example other active agents, initiators,accelerators, hardeners, stabilisers, antioxidants, adhesion promoters,fillers and the like may be incorporated within the polymer. Additionalmaterials(s) may be mixed with the polymer before or after contactingwith deposition matter, or may be introduced by subsequent soaking orimpregnation of the product composite having internally distributeddeposition matter.

If it is desired to introduce an adhesion promoter into the polymercomposite, the promoter may be used to impregnate or coat particles ofdeposition matter prior to introduction into the polymer composite, bymeans of simple mixing, spraying or other known coating steps, in thepresence or absence of fluid as hereinbefore defined. Preferably coatingis performed in conjunction with mixing with fluid as hereinbeforedefined whereby excellent coating is obtained. For example the adhesionpromoter is dissolved in fluid as hereinbefore defined and the solutionis contacted with particles of deposition matter as hereinbeforedefined. Alternatively the adhesion promoter is introduced into theautoclave during the mixing and/or polymerisation step whereby itattaches to particles of deposition matter in desired manner.

Preferably the total amount of fillers including the deposition matterlies in the region of 0.01-99.9 wt %, preferably 0.1-99 wt %, morepreferably in excess of 50 or 60 wt %, up to for example 70 or 80 wt %.

In some cases it may be desirable to introduce an initiator oraccelerator to initiate (partial) curing prior to and/or subsequent torelease of fluid, and initiation may be simultaneous with introductionor may be delayed, activated by increase in temperature. Alternatively aspray drying step may be employed in place of the curing step prior toor simultaneously with release of the fluid. In this case a post-curingmay be employed. This may have advantages in terms of ease ofmanufacturing and simplicity of apparatus employed.

In a further aspect of the invention there is provided a polymercomposite obtained with the process of the invention as hereinbeforedefined.

In a further aspect of the invention there is provided a polymercomposite comprising a porous or non porous polymer throughout whichparticulate deposition matter as hereinbefore defined is distributedwith desired uniformity, preferably with high uniformity in excess of80% for example in excess of 98%. In a particular advantage thecomposite comprises exceedingly low levels of deposition matter of theorder of picograms or nanograms per 5 g polymer, or presented asconcentration of deposition matter on polymer, in low volumes in therange 1×10¹ to 1×10³ ng/mg at excellent levels of uniformity and batchreproducibility, and/or of very low particle size of the order of 10microns, 1 micron or 0.1 microns.

In a further advantage, contrasted with other methods of encapsulating(e.g. double emulsion) and introducing biological material which giverise to relatively large particles which give an uneven release withtime, the process of the present invention enables internallydistributing very small particles of deposition matter thus giving amuch even release profile (reduced burst phase effect). Moreover thecomposite of the invention has been found to give release over a periodof several months, and this is in contrast to the corresponding surfacedeposited polymer which may lose its surface deposit over the course ofdays.

The composite of the invention may be distinguished from prior artcomposite prepared by simple impregnation techniques and those of WO91/09079 which show agglomeration of impregnation matter etc.

Advantageously it has been found that very low and very high loading maybe obtained according to the process of the present invention, which isnot possible with known processes, by virtue of the uniform morphologyof polymer and deposition matter, and loadings of deposition matter inthe range from 1×10⁻¹²-99.9 wt %, for example in the region 1×10⁻¹² to1×10⁻⁹ wt %, midrange of from 20 to 50 wt % or in excess of 50 wt %, orin excess of 80 wt % may be obtained.

The polymer composite may be in desired form suitable for thehereinbefore mentioned uses. Suitably the composite may be obtained ingranular or monolith form and is preferably in monolith form for use asa scaffold or drug delivery device.

For use as bioremediation, biocatalyst or biobarrier for human or animalor plant matter, the composite may be in a suitable shaped form or maybe impregnated into a shaped product, to provide a barrier film,membrane, layer, clothing or sheet.

For use as a structural component, for example comprising the polymerand optional additional synthetic or natural metal, plastic, carbon orglass fibre mesh, scrim, rod or like reinforcing for medical or surgicalinsertion, the composite may be adapted for dry or wet insertion into adesired host structure, for example may be in powder, pellet, granule ormonolith form suited for insertion as a solid monolith into bone ortissue, as fillers or cements for wet insertion into bone or teeth or assolid aggregates or monoliths for orthopaedic implants such as pins, ordental implants such as crowns etc. Insertion may be by injection,surgical insertion and the like.

The polymer composite may be of any desired particle size in the rangeof 0.1 or 1 micron powders, preferably from 50 or 200 micron for usewith larger particle size deposition matter up to monoliths of the orderof 20 centimetres magnitude. It is a particular advantage of the presentinvention that the polymer composite is obtained in the desired form inuniform size particles such as powder, pellets and the like. Accordinglyif it is desired to obtain a random or discrete distribution of particlesize the polymer composite may be milled or may be blended fromdifferent size batches.

Composite particle size may be controlled according to known techniquesby controlled removal of plasticising fluid. If it is desired to obtainparticulate composite, the process mixture is suitably removed from themixing chamber under plasticising conditions into a separate containerunder ambient conditions through a nozzle or like orifice of desiredaperture, and under desired difference of conditions and removal rate,adapted to provide the desired particle size. Spray drying apparatus andtechniques may commonly be employed for the technique.

If it is desired to obtain a polymer composite in the form of monoliths,the plasticising fluid is suitably removed using known techniques forfoaming polymers. Accordingly the polymer mix is retained in thereaction vessel and conditions are changed from plasticising to ambientat a desired rate to cause removal of the fluid from the polymer mix.Depending on the nature of the polymer it is possible to obtain themonolith in porous foamed state if desired, having interconnecting poresand channels created by the removal of the plasticising fluid, simply byselecting a polymer consistency which is adapted to retain its foamedstate.

Monoliths may be formed into desired shape during the processingthereof, for example by removal of plasticising fluid from a mixingvessel, or from a mould internal to mixing vessel having the desiredmonolith shape. Alternatively monolith may be removed from the mixingvessel and cut to desired shape or transferred directly into a mould.

In a further aspect of the invention there is provided a scaffoldcomprising a polymer composite having internally distributed depositionmatter as hereinbefore defined, suitably sized and shaped for a desiredapplication as hereinbefore defined.

A scaffold according to the invention is suitably in the form of asurgical implant, synthetic bone composite, organ module, biocatalystfor remediation or synthesis, or the like. The scaffold may bebiodegradable or otherwise, for biodegradation in the body and ingrowthby native cells, or for biodegradation in the environment aftercompletion of bioremediation avoiding in each case the need forsubsequent operation to remove the polymer.

In a further aspect of the invention there is provided an apparatus foruse in the preparation of a polymer composite as hereinbefore defined.Suitably the apparatus comprises one or more pressure vessels adaptedfor temperature and pressure elevation and comprising means for mixingthe contents. The pressure vessel may include means for depressurisationor for discharging of contents into a second pressure vessel at lowerpressure. The apparatus comprises means for introduction of polymer,deposition matter and plasticising fluid and any other materials whilstthe vessel is pressurised, as commonly known in the art.

In a further aspect of the invention there is provided a polymercomposite as hereinbefore defined or a scaffold thereof for use as asupport or scaffold for drug delivery, for use in bioremediation, as abiocatalyst or biobarrier for human or animal or plant matter, for useas a structural component, for example comprising the polymer andoptional additional synthetic or natural metal, plastic, carbon or glassfibre mesh, scrim, rod or like reinforcing for medical or surgicalinsertion, for insertion as a solid monolith into bone or tissue, asfillers or cements for wet insertion into bone or teeth or as solidaggregates or monoliths for orthopaedic implants such as pins, or dentalimplants such as crowns etc.

The invention is now illustrated in non limiting manner with referenceto the following examples and Figures wherein.

FIG. 1A-D shows scanning electron micrograph images of compositesfabricated by the process of WO 98/51347 (Howdle et al) employed in thepresent invention; in Images A and B of an internal fracture surface ofa monolith composite of calcium hydroxyapatite (40 wt %) and PLGA (60 wt%), at low magnification the distribution of calcium hydroxyapatitethroughout the matrix and the production of pores is evident, at highermagnification the intimate mixing of guest particles and polymer isobserved; in image C catalase (50% wt) is shown incorporated into a PLGAmatrix (50%), micron scale pores in the polymer and the distinctiveprotein particle morphology are evident; in image D a high surface areamicroparticle composite (fluorescein (sodium salt) (8 wt %) andpolycaprolactone (92 wt %)) are observed produced by direct atomisation,ie after fast depressurisation through an orifice.

FIGS. 2 and 3 show scanning electron micrograph images and correspondingmercury porosimetry data for PLA composites fabricated by the process ofWO 98/51347 (Howdle et al) employed in the present invention withcontrol of PLA pore structure by changing de-pressurisation conditions;in FIG. 2 the image shows presence of a small population of large poresobtained by de-pressurisation over a 2-hour period (“slow”); in FIG. 3the image shows an increase in porosity and a more heterogeneousdistribution obtained by de-pressurisation over a 2-minute period(“fast”); data obtained by mercury porosimetry demonstrate that finecontrol over micropore distribution is achieved by changing only thede-pressurisation rate, with “slow” depressurisation creating pores inthe 50 to 500 nm range, whilst “fast” depressurisation is strikinglydifferent and creates pores in the 500 nm to 5 μm range.

FIG. 4 shows a schematic of the method of the invention in whichfluorescent protein solution is adsorbed onto the polymer surface, theprotein is confined to the surface and does not penetrate the bulk;confocal cross section through the polymer from the top surface showsprotein confined to the edge and outer pores of the PLA scaffold;thereafter the polymer: protein complex is plasticised in CO₂, theprotein is shown distributed throughout the sample, and the resultingfluorescence is homogeneous with the protein redistributed from thesurface to the bulk of the polymer.

FIG. 5 shows recovery of protein activity after double processing in CO₂FIG. 6 shows protein release with time for the composite of FIG. 4 andcomparative composite not according to the invention.

METHODS AND MATERIALS

Cell Culture

Bone marrow samples (16 patients in total: 11 females and 5 males aged14-83, with a mean age of 63.8 years) were obtained from patientsundergoing routine total hip replacement surgery. Only tissue, whichwould have been discarded, was used with ethical approval. Human bonemarrow cells were cultured on poly(lactic acid) porous scaffoldsencapsulated with and without recombinant human BMP-2 or PLA scaffoldsadsorbed with rhBMP-2. In vitro assays included human bone marrow cellswith or without addition of recombinant human BMP-2 (50 ng/ml) in basal(10% αMEM) and osteogenic conditions (10% αMEM supplemented with 100μg/ml ascorbate and 10 nM dexamethasone).

Chorioallantoic Membrane Assay

Fertilised eggs were incubated for 10-18 days using a Multihatchautomatic incubator (Brinsea Products, Sandford, UK) at 37° C. in ahumidified atmosphere. Chick femurs were excised from day 18 chickembryos and a wedge-shaped segmental defect created in the middle of thefemur, into which the scaffold construct was placed to fill the defectsite. Chick bone and scaffolds (29 samples) were placed directly ontothe CAM of 10-day-old eggs (through a 1 cm² square section cut into theshell) and incubation continued for a further 7 days. Thefemoral/scaffold explant was then placed onto the CAM and incubation, at37° C., continued for a further 7 days. Explants were then harvested andthe chick embryo killed by decapitation. Prior to histochemicalanalysis, scaffold and explant samples were then fixed in 95% ethanol,processed to paraffin wax and 5 μm sections prepared for histology.

EXAMPLE 1 Preparation of Polymer Material

Poly(DL-lactic acid) (Alkermes Medisorb, low I.V. Mw=85 kD,polydispersity=1.4) was ground to a fine grain size powder in a pestleand mortar. Alternatively, particles were produced by forcing thepoly(DL-lactic acid) out of a vessel pressurized with CO₂ through anorifice. The particles were retrieved from a cyclone collector, the CO₂may be repressurised and recycled. The methodology is based on theantisolvent technique of particle generation from supercriticalsuspension (PGSS).

The polymer may also be prepared as a highly porous monolith usingsupercritical fluid processing. In this case porous scaffolds wereprepared in moulds prepared from 48-well tissue culture plates (Costar,USA). 12×100 mg (+1 mg) PLA were weighed out into the wells, and themould was sealed inside the autoclave. The autoclave was heated to 35°C. before filling with CO₂ over a period of 30 minutes to a pressure of207 Bar. This long filling time minimised the potentially detrimentaleffects of excessive Joule-Thompson heating on the biologically activesubstrate as the system was pressurised. The plasticising CO₂-polymermixture was allowed to equilibrate for 20 minutes before venting toatmospheric pressure over 8 minutes. The pressure was controlledthroughout the preparation using a JASCO BP-1580-81 programmablebackpressure regulator. The autoclave temperature remained below 38° C.throughout the filling step, and the flow rate of CO₂ during theequilibration step was 12 cm³ min⁻¹. After the CO₂ processing, the mouldcontaining the foamed polymer was removed from the autoclave and theresidual gas allowed to escape for 2 hours.

EXAMPLE 2 Addition of the Biological Material—Protein

The protein, in this example avidin tagged with the fluorescent moleculerhodamine (Sigma), was dissolved in distilled water to give solutions ata concentration of 1 microgram and 10 microgram per ml in water). Theliquid may alternatively be chosen from any liquid that dissolves thebiological molecule but does not dissolve the polymer. 0.5 cm³ aliquotsof protein solution were pipetted onto approx 250 mg samples of polymermaterial and remained in contact with the samples for a period ofbetween 1 sec and 48 hours. During this exposure, a freeze dryingprocess was used to remove the liquid. We have freeze-dried a range ofavidin-rhodamine and ribonuclease solutions (1 microgram −250 mg/ml)onto both porous scaffolds and polymer powders for periods of up to 48hours. Control scaffolds without any protein addition were prepared.

Confocal fluorescence microscopy of this material confirmed that theavidin rhodamine was confined to the surface of the polymer material andwas not distributed with the solid mass of the polymer (FIG. 4).

EXAMPLE 3 Re-Distribution of the Biological Material—Protein

One scaffold from each protein concentration sample from Example 2, wasremoved from the well to act as control. The remaining examples wereplaced into a high pressure autoclave and heated to 35° C.,replasticised in CO₂ using the same procedure as Example 2 above. FIG. 4shows a schematic of the plasticising process. Confocal fluorescencemicroscopy of this re-processed material showed that the avidinrhodamine was re-distributed within the bulk of the polymer (FIG. 4).Confocal microscopy was performed using a Leica TCS4D system with aLeica DMRBE upright fluorescence microscope and an argon-krypton laser.The red fluorescence of TRITC Avidin-Rhodamine was excited with the 568nm laser line.

EXAMPLE 4 Addition of Biological Material—Enzyme

To prove that the activity of biological material was unaffected by thistreatment, 100 microlitres of 250 mg/ml of the enzyme ribonuclease A(Sigma) was adsorbed onto 8 batches of 100 mg poly(DL-lactic acid)powder using the method of the above Examples and freeze-dried for48-hours.

EXAMPLE 5 Redistribution of Biological Material—Enzyme

The powder of Example 4 was processed using the conditions in Example 3to produce polymer foam composites.

EXAMPLE 6 Evidence for Retention of Activity

The ribonuclease enzyme was released from the foams obtained in Example5 in a Tris buffer (pH 7.13) at physiological temperatures. Using aspecific ribonuclease substrate, cytidine-2′:3′-monophospate, therecovery of activity was monitored by the conversion of the substrate toa form that could be detected by a UV spectrophotometer (Table 1). Fullbiological activity of the protein was retained.

Results

FIG. 4 shows a schematic of the supercritical fluid process.Concentration profiles of the fluorescent avidin-rhodamine complex areshown after the freeze-drying step and after plasticising CO₂reprocessing. Following the initial freeze-drying, fluorescence islocalised at the exposed surfaces of the scaffold, i.e. the top surfaceand the walls of pores. After CO₂ reprocessing, the complex isdistributed throughout the sample, and the resulting fluorescence ishomogeneous.

The schematic is supported by data from confocal microscopy. On the leftare eight images that follow the edge of a pore in a sample from the topsurface to a depth of 77.4 μm after the initial freeze-drying step. Theimages show a decreasing intensity of fluorescence as the distance fromthe top surface increases, except for a narrow region localised at theedge of the pore.

The series on the right depicts a sample that has been reprocessed inplasticising CO₂. Here again, the series follows the edge of a pore to adepth of 82.5 μm below the surface. In contrast to the unprocessedscaffold, fluorescence is observed throughout the scaffold withappreciable intensity seen both in the bulk and at the pores' surface.

Ribonuclease activity was measured after release into Tris buffersolution from scaffolds after processing in scCO₂ (FIG. 5). The rate ofreaction of conversion of cytidine-2′,3′-monophosphate tocytidine-3′-phosphate was measured by the change in absorbance at 284nm. The black circles (samples) represent the activity of the enzymecompared to the standards (open circles). The mean recovery of activitywas 100.8% (+9.8%) indicating that enzyme activity is Actual MaximalPercentage Sam- Amount RN Rate Actual Rate Standard Recovery ple(microgram) (dA 284 nm) (dA 284 nm) Deviation (%) 1 66 0.0354 0.03340.0017 94.4 2 69 0.0374 0.0397 0.0012 106.2 3 71 0.0384 0.0333 0.002486.8 4 60 0.0323 0.0309 0.0021 95.5 5 50 0.0270 0.0295 0.0021 109.4 6 640.0345 0.0339 0.0048 98.3 7 62 0.0334 0.0329 0.0026 98.4 8 38 0.02050.0241 0.0034 117.4retained throughout the process. The correlation between sample andstandard activity is high (R²=0.9959).

EXAMPLE 7 Evidence of Controlled Release

FIG. 6 displays the protein release behaviour from Example 6 as afunction of time. Where the protein has been dried onto the polymerscaffold without a second plasticising CO₂ processing step, the proteinis released very quickly with nothing remaining after two days (Blacktriangles). In samples which have been subjected to the SCF reprocessingstep, the release is far more protracted. After an initial “burst” phase(0-1 days), the rate of release stabilises for approximately three weeksbefore degradation of the polymer matrix allows the protein to escape.The profile then follows a rectilinear relationship until the exhaustionof the protein after approximately 80 days.

EXAMPLE 8 Addition of Biological Material—Growth Factor

Scaffold Generation and rhBMP-2 Encapsulation

Polymer obtained as in Example 1 was loaded with the Growth Factorrecombinant human bone morphogenetic protein-2 (rhBMP-2). Poly(DL-lacticacid) and rhBMP-2 (100 ng/mg PLA) were mixed together using acombination of conventional solution and supercritical carbon dioxideprocessing to generate porous (50-200 μm) scaffolds. Recombinant BMP-2was adsorbed onto poly(D,L-lactic acid) powder (Alkermes Inc., USA; lowinherent viscosity, Mw 84 kDa, polydispersity=1.4) at a concentration of100 ng/mg polymer. The polymer:protein mixture was processed using asupercritical carbon dioxide pressurized to 207 bar and heated to 35° C.for 20 minutes in a high pressure vessel. Upon depressurization, theprotein is encapsulated within the polymer and pores are formed in thepolymer matrix by the escape of the carbon dioxide gas. Functionallyactive recombinant human BMP-2 was derived from E. Coli, at greater than98% purity in a largely homogenous form. In this procedure, theefficient processing of the liquefied polymer in scCO₂ at near ambienttemperatures results in a homogeneous distribution of the bioactivefactor throughout the polymer matrix. These mild processing conditionsallow the processing of growth factors that are heat or solventsensitive without further degradation or damaging their biologicalactivity.

EXAMPLE 9 Cell Growth in PLA

Human bone marrow cell/PLA constructs were cultured in 10% FCS αMEMsupplemented with osteogenic medium containing 5 mM inorganic phosphatefor the final 48 hours of the culture period and mineralization wasdetected by von Kossa staining.

Histochemistry and Immunocytochemistry

Prior to histochemical analysis, PLA scaffold samples were fixed with 4%Paraformaldehyde or 95% ethanol, dependent on the staining protocol and,as appropriate, processed to paraffin wax and 5 μm sections prepared.Negative controls were included in all studies. i) Alkaline phosphataseactivity: Cultures stained using the Sigma alkaline phosphatase kit(no.85) according to the manufacturer's instructions; ii) Alcianblue/Sirius red: Samples were stained using Weigert's haematoxylin, 0.5%alcian blue (in 1% acetic acid) and sirius red (in saturated Picricacid). iii) Toluidine Blue and Von Kossa Staining: Samples were stainedwith 1% AgNO3 under UV light for 20 minutes until black deposits werevisible and after air drying, slides were counterstained with toluidineblue.

C2C12 Alkaline Phosphatase Assay

BMP-2 has the ability to induce C2C12 promyoblast differentiation intothe osteoblast lineage^((33,34,35)). After encapsulation of 0.01% (w/w)rhBMP-2 within PLA scaffolds, the bioactivity of rhBMP-2 released fromthe polymer was determined using C2C 12 cells. Briefly, human bonemarrow stromal cells were cultured in the presence or absence of rhBMP-2encapsulated PLA scaffold, or passaged onto rhBMP-2 encapsulated PLAscaffold or PLA scaffold alone in 10% FCS DMEM at 37° C. and 5% CO₂ forthree days. Samples were fixed in ethanol and stained for alkalinephosphatase.

Bioactivity of rhBMP-2 Encapsulated PLA Scaffolds

After encapsulation of rhBMP-2 within PLA scaffolds (100 ng/mg PLA), thebioactivity of rhBMP-2 released from PLA scaffolds was determined usinginduction of the C2C 12 promyoblast cell line into the osteogeniclineage as detected by alkaline phosphatase expression. Alkalinephosphatase-positive cells were observed following culture of C2C12cells in presence of or on rhBMP-2 encapsulated PLA scaffolds (FIGS. 1A,C). No induction of alkaline phosphatase-positive cells was observedusing blank scaffolds (FIGS. 1B, D). As expected, rhBMP-2 (50 ng/ml)adsorbed on PLA promoted human bone marrow stromal cell adhesion,spreading, proliferation, and differentiation on PLA porous scaffold invitro as observed by SEM, confocal microscopy and expression of type Icollagen histochemistry (data not shown).

Human Osteoprogenitor Growth on rhBMP-2 Encapsulated Scaffolds

Following demonstration of the ability of using rhBMP-2 encapsulated PLAscaffold to stimulate differentiation of C2C 12 promyoblast towards theosteoblast lineage, the potential of rhBMP-2 scaffolds to inducedifferentiation and mineralisation of human bone marrow stromal cellswas examined in vitro and in vivo.

i) CAM Culture

Culture of human osteoprogenitors on rhBMP-2 encapsulated PLA scaffoldson the chick chorioallantoic membrane model showed that encapsulatedrhBMP-2 stimulated human bone marrow stromal cell growth anddifferentiation in the PLA scaffolds (FIGS. 2B-D). Extensiveangiogenesis, as evidenced by new blood vessel growth, was observed onthe scaffold/cell constructs from the CAM to the implanted constructover a period of 7 days (FIG. 2A). New cartilage and bone were observedwithin the chick bone defect as detected by alcian blue and sirius redstaining (FIGS. 2B, C) and the use of polarized light microscopy todemonstrate collagen birefringence within the newly formed matrix (FIG.2D).

Subcutaneous Implantation

Confluent primary human bone marrow cells were trypsinised and seeded(2×10⁵ cells/sample in serum free αMEM) onto PLA scaffolds adsorbed withrhBMP-2 or rhBMP-2 encapsulated PLA scaffolds for 15 hours. Blank (PLAalone) scaffolds were set up in the absence of cells. After 15 hours,constructs were placed in osteogenic media for a further 3 days, priorto subcutaneous implantation into MF1-nu/nu mice (20-24 g, 4-5 weeksold) as previously described⁽³⁶⁾. After 4-6 weeks, the mice were killedand specimens were collected and fixed in 95% ethanol for histochemicalanalysis.

ii) Subcutaneous Implant Model

Primary human bone marrow cells were seeded onto PLA scaffoldsencapsulated with rhBMP-2 and subcutaneous implanted (8 samples) in nudemice for 6 weeks (PLA alone served as a negative control). Poor cellgrowth and negligible bone matrix synthesis was observed on PLAscaffolds alone (in the absence of rhBMP-2) implanted in nude mice withonly fibrous tissue and adipose tissue observed (FIG. 3E). In contrast,rhBMP-2 encapsulated scaffolds promoted human bone marrow stromal celladhesion, proliferation, differentiation with extensive evidence of newbone matrix deposition as detected by Alcian blue/Sirius red stainingfor cartilage and bone respectively (FIGS. 3A and 3B). Furthermore,evidence of organised new woven bone within the encapsulated constructswas confirmed by birefringence of collagen using polarized microscopy(FIG. 3B). The efficacy of rhBMP-2 to induce bone formation wasconfirmed by HBM cell in-growth and bone matrix formation into rhBMP-2adsorbed PLA scaffolds as detected by Alcian blue and Sirius redstaining (FIG. 3C) and (FIG. 3D) Type I collagen staining. Only fibroustissue and fat tissue were observed in blank (PLA alone) scaffolds (FIG.3E).

Intra-Peritoneal Implantation

The diffusion chamber (130 μl capacity) model provides an enclosedenvironment within a host animal to study the osteogenic capacity ofskeletally derived cell populations, which resolves the problems of hostversus donor bone tissue generation. Cells were recovered by collagenase(Clostridium histolyticum, type IV; 25 U/ml) and trypsin/EDTA digestion.Human bone marrow cells were sealed in diffusion chambers (2×10⁶cells/chamber) together with PLA porous scaffold encapsulated oradsorbed with or without rhBMP-2. Chambers were implantedintra-peritoneally in MF1-nu/nu mice and after 10 weeks the mice werekilled, chambers were removed and examined by X-ray analysis prior tofixation in 95% ethanol at 4° C. Polymer samples were processedundecalcified and sectioned at 5 μm and stained for toluidine blue, typeI collagen, osteocalcin and mineralisation by von Kossa.

iii) Diffusion Chamber Model

Recombinant human BMP-2 encapsulated PLA scaffolds seeded with humanosteoprogenitor cells, showed morphologic evidence of new bone andcartilage matrix formation as examined by Alcian blue and Sirius redstaining (FIGS. 3G, 3J) and by X-ray analysis (FIG. 31) after 10 weeksimplantation within diffusion chambers. Metachromatic staining wasobserved using toluidine blue and collagen deposition and new matrixsynthesis was confirmed by birefringence microscopy (FIG. 3H). Cartilageformation could be observed within rhBMP-2 encapsulated PLA scaffoldsconfirming penetration of human osteoprogenitors through the scaffoldconstructs (FIG. 3J). No bone formation was observed on cell/PLAscaffold constructs alone (FIG. 3F).

Further aspects and advantages of the invention will be apparent fromthe foregoing.

1-24. (canceled)
 25. A process for the preparation of a polymercomposite comprising internally distributed deposition matter whereinthe process comprises providing a deposit of deposition matter at thesurface of a solid state polymer substrate by fluid phase deposition ofdiscrete particles or dissolved deposition matter by immersion orspraying of solid state polymer substrate with a solution, dispersion orsuspension of deposition matter, drying by freezing, evaporation,heating or blotting whereby the deposition matter adsorbs from liquidphase on to the polymer surface and forms an adsorption layer ofdeposition matter which is intact to solvent and impact effectscontacting the surface deposited polymer with a plasticising fluid or amixture of plasticising fluids under plasticising conditions toplasticise and/or swell the polymer and internally distribute depositionmatter, and releasing the plasticising fluid or fluids to obtain polymercomposite.
 26. A process as claimed in claim 25 wherein depositionmatter is present, presented as concentration of deposition matter onpolymer, in the range 1×10¹ to 1×10³ ng/mg, or of the order of picogramsor nanograms per 5 g polymer, or 1×10⁻¹² to 1×10⁻⁹ wt %.
 27. A processas claimed in claim 25 which comprises providing a deposit at thesurface of a high surface area polymer substrate.
 28. Process as claimedin claim 25 wherein the polymer substrate comprises a powder bed or ahigh porosity matrix.
 29. A process as claimed in claim 25 wherein adeposit comprises a deposition layer of deposition matter on anyinternal and external exposed surfaces of the polymer substrate,including any exposed surface pores; over the entire surface area oronly part or parts thereof.
 30. A process as claimed in claim 25 whereinthe solid state polymer substrate is obtained by contacting polymer withplasticising fluid or a mixture of plasticising fluids underplasticising conditions to plasticise the polymer, and releasing thefluid in manner to obtain a solid state substrate polymer.
 31. A processas claimed in claim 25 carried out in the absence of additional solventcapable of dissolving the deposition matter.
 32. A process as claimed inclaim 25 wherein immersion is for a time of the order 1 second up to 48hours.
 33. A process as claimed in claim 25 wherein drying is for a timeup to 48 hours.
 34. A process as claimed in claim 25 whereinplasticising conditions comprise a temperature in the range −200° C. to+500° C.
 35. A process as claimed in claim 25 wherein plasticisingconditions comprise a pressure from in excess of 1 bar to 10000 bar. 36.A process as claimed in claim 25 wherein the process is carried out fora contact time of surface deposited polymer and plasticising fluid of 20milliseconds up to 5 minutes.
 37. A process as claimed in claim 25 whichis carried out without blending.
 38. A process as claimed in claim 25wherein plasticising fluid is selected from carbon dioxide, di-nitrogenoxide, carbon disulphide, aliphatic C₂₋₁₀ hydrocarbons such as ethane,propane, butane, pentane, hexane, ethylene, and halogenated derivativesthereof such as for example carbon tetrafluoride or chloride and carbonmonochloride trifluoride, and fluoroform or chloroform, C₆₋₁₀ aromaticssuch as benzene, toluene and xylene, C₁₋₃ alcohols such as methanol andethanol, sulphur halides such as sulphur hexafluoride, ammonia, xenon,krypton, and mixtures thereof.
 39. A process as claimed in claim 25wherein deposition material is selected from (pharmaceutical) drugs andveterinary products; agrochemicals as pest and plant growth controlagents; human and animal health products; human and animal growthpromoting, structural, or cosmetic products including products intendedfor growth or repair or modelling of the skeleton, organs, dentalstructure; absorbent biodeposition materials for poisons, toxins.
 40. Aprocess as claimed in claim 25 wherein deposition matter alternativelyor additionally comprises function enhancing components, includingnaturally occurring or synthetic or otherwise modified growth promoters,biocompatibilisers, vitamins, proteins, glycoproteins, enzymes, nucleicacid, carbohydrates, minerals, nutrients, steroids, ceramics and thelike and functioning matter such as spores, viruses, mammalian, plantand bacterial cells.
 41. Process as claimed in claim 25 wherein polymeris selected from: polyesters including poly(lactic acid), poly(glycolicacid), copolymers of lactic and glycolic acid, copolymers of lactic andglycolic acid with poly(ethylene glycol), poly(e-caprolactone),poly(3-hydroxybutyrate), poly(p-dioxanone), poly(propylene fumarate);poly (ortho esters); polyanhydrides; Poly(amino acids); polyacetals;polyketals; polyorthoesters; Polyphosphazenes; azo polymers; syntheticNon-biodegradable Polymers selected from: Vinyl polymers includingpolyethylene, poly(ethylene-co-vinyl acetate), polypropylene, poly(vinylchloride), poly(vinyl acetate), poly(vinyl alcohol) and copolymers ofvinyl alcohol and vinyl acetate, poly(acrylic acid) poly(methacrylicacid), polyacrylamides, polymethacrylamides, polyacrylates,Poly(ethylene glycol), Poly(dimethyl siloxane), Polyurethanes,Polycarbonates, Polystyrene and derivatives; and Natural Polymersselected from carbohydrates, polypeptides and proteins.
 42. A processfor the preparation of a polymer composite comprising internallydistributed deposition matter wherein the process comprises providing adeposit of deposition matter at the surface of a solid state polymersubstrate by fluid phase deposition of discrete particles or dissolveddeposition matter by immersion or spraying of solid state polymersubstrate with a solution, dispersion or suspension of depositionmatter, drying by freezing, evaporation, heating or blotting or by solidphase deposition by powder coating, dusting, rolling or adheringcontacting the surface deposited polymer with a plasticising fluid or amixture of plasticising fluids under plasticising conditions toplasticise and/or swell the polymer and internally distribute depositionmatter, and releasing the plasticising fluid or fluids to obtain polymercomposite wherein deposition matter is present, presented asconcentration of deposition matter on polymer, in the range 1×10¹ to1×10³ ng/mg, or of the order of picograms or nanograms per 5 g polymer,or 1×10⁻¹² to 1×10⁻⁹ wt %.
 43. A process as claimed in claim 42 whichcomprises providing a deposit at the surface of a high surface areapolymer substrate.
 44. Process as claimed in claim 42 wherein thepolymer substrate comprises a powder bed or a high porosity matrix. 45.A process as claimed in claim 42 wherein a deposit comprises adeposition layer of deposition matter on any internal and externalexposed surfaces of the polymer substrate, including any exposed surfacepores; over the entire surface area or only part or parts thereof.
 46. Aprocess as claimed in claim 42 wherein the solid state polymer substrateis obtained by contacting polymer with plasticising fluid or a mixtureof plasticising fluids under plasticising conditions to plasticise thepolymer, and releasing the fluid in manner to obtain a solid statesubstrate polymer.
 47. A process as claimed in claim 42 carried out inthe absence of additional solvent capable of dissolving the depositionmatter.
 48. A process as claimed in claim 42 wherein immersion is for atime of the order 1 second up to 48 hours.
 49. A process as claimed inclaim 42 wherein drying is for a time up to 48 hours.
 50. A process asclaimed in claim 42 wherein plasticising conditions comprise atemperature in the range −200° C. to +500° C.
 51. A process as claimedin claim 42 wherein plasticising conditions comprise a pressure from inexcess of 1 bar to 10000 bar.
 52. A process as claimed in claim 42wherein the process is carried out for a contact time of surfacedeposited polymer and plasticising fluid of 1 millisecond up to 5 hours.53. A process as claimed in claim 42 which is carried out withoutblending.
 54. A process as claimed in claim 42 wherein plasticisingfluid is selected from carbon dioxide, di-nitrogen oxide, carbondisulphide, aliphatic C₂₋₁₀ hydrocarbons such as ethane, propane,butane, pentane, hexane, ethylene, and halogenated derivatives thereofsuch as for example carbon tetrafluoride or chloride and carbonmonochloride trifluoride, and fluoroform or chloroform, C₆₋₁₀ aromaticssuch as benzene, toluene and xylene, C₁₋₃ alcohols such as methanol andethanol, sulphur halides such as sulphur hexafluoride, ammonia, xenon,krypton, and mixtures thereof.
 55. A process as claimed in claim 42wherein deposition material is selected from (pharmaceutical) drugs andveterinary products; agrochemicals as pest and plant growth controlagents; human and animal health products; human and animal growthpromoting, structural, or cosmetic products including products intendedfor growth or repair or modelling of the skeleton, organs, dentalstructure; absorbent biodeposition materials for poisons, toxins.
 56. Aprocess as claimed in claim 42 wherein deposition matter alternativelyor additionally comprises function enhancing components, includingnaturally occurring or synthetic or otherwise modified growth promoters,biocompatibilisers, vitamins, proteins, glycoproteins, enzymes, nucleicacid, carbohydrates, minerals, nutrients, steroids, ceramics and thelike and functioning matter such as spores, viruses, mammalian, plantand bacterial cells.
 57. Process as claimed in claim 42 wherein polymeris selected from: polyesters including poly(lactic acid), poly(glycolicacid), copolymers of lactic and glycolic acid, copolymers of lactic andglycolic acid with poly(ethylene glycol), poly(e-caprolactone),poly(3-hydroxybutyrate), poly(p-dioxanone), poly(propylene fumarate);poly(ortho esters); polyanhydrides; Poly(amino acids); polyacetals;polyketals; polyorthoesters; Polyphosphazenes; azo polymers; syntheticNon-biodegradable Polymers selected from: Vinyl polymers includingpolyethylene, poly(ethylene-co-vinyl acetate), polypropylene, poly(vinylchloride), poly(vinyl acetate), poly(vinyl alcohol) and copolymers ofvinyl alcohol and vinyl acetate, poly(acrylic acid) poly(methacrylicacid), polyacrylamides, polymethacrylamides, polyacrylates,Poly(ethylene glycol), Poly(dimethyl siloxane), Polyurethanes,Polycarbonates, Polystyrene and derivatives; and Natural Polymersselected from carbohydrates, polypeptides and proteins.
 58. A polymercomposite when obtained by the process of claim
 25. 59. The use of apolymer composite or a scaffold thereof prepared by the process of claim25, for drug delivery, in bioremediation, as a biocatalyst or biobarrierfor human or animal or plant matter, as a structural componentcomprising the polymer and optional additional synthetic or naturalmetal, plastic, carbon or glass fibre mesh, scrim, rod or likereinforcing for medical or surgical insertion, for insertion as a solidmonolith into bone or tissue, as fillers or cements for wet insertioninto bone or teeth or as solid aggregates or monoliths for orthopaedicimplants such as pins, or dental implants such as crowns.